scholarly journals Genetic analysis of the Epstein–Barr virus-coded leader protein EBNA-LP as a co-activator of EBNA2 function

2001 ◽  
Vol 82 (12) ◽  
pp. 3067-3079 ◽  
Author(s):  
Eamon M. McCann ◽  
Gemma L. Kelly ◽  
Alan B. Rickinson ◽  
Andrew I. Bell
Keyword(s):  
Genetic Analysis ◽  
Epstein Barr Virus ◽  
Cell Transformation ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Lmp1 Expression ◽  
Leader Protein ◽  
Epstein Barr ◽  
The Individual ◽  
Unique Domain

Co-operation between the Epstein–Barr virus (EBV)-coded leader protein EBNA-LP and the nuclear antigen EBNA2 appears to be critical for efficient virus-induced B cell transformation. Here we report the genetic analysis of EBNA-LP function using two transient co-transfection assays of co-operativity, activation of latent membrane protein 1 (LMP1) expression from a resident EBV genome in Akata-BL cells and activation of an EBNA2-responsive reporter construct. Small deletions were introduced into each of five conserved regions (CRs) of EBNA-LP sequence present in type 1 and type 2 EBV strains and in several primate lymphocryptovirus EBNA-LP homologues. Deletions within all three CRs in the EBNA-LP W1W2 repeat domain completely abrogated function, through inhibition of nuclear localization in the cases of CR1 and CR2 but not of CR3; deletions within CR4 and CR5 in the Y1Y2 unique domain had relatively little effect, yet loss of the whole Y2 sequence blocked activity. Alanine substitution of serine residues within potential phosphorylation sites identified two mutants of particular interest. Substitution of three such residues (S34,36,63) within W1W2 not only abrogated EBNA-LP activity but was associated with a complete loss of EBNA2 detectability in co-transfected cells, implying possible destabilization of the co-expressed EBNA2 protein. More importantly the individual substitution of S36 completely blocked EBNA-LP/EBNA2 co-operativity while retaining EBNA2 expression. We infer critical roles for the CR3 domain and for the S36 residue in EBNA-LP’s co-operative function.

scholarly journals Conserved Region CR2 of Epstein-Barr Virus Nuclear Antigen Leader Protein Is a Multifunctional Domain That Mediates Self-Association as well as Nuclear Localization and Nuclear Matrix Association

2002 ◽  
Vol 76 (3) ◽  
pp. 1025-1032 ◽  
Author(s):  
Michiko Tanaka ◽  
Akihiko Yokoyama ◽  
Mie Igarashi ◽  
Go Matsuda ◽  
Kentaro Kato ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nuclear Localization ◽  
Nuclear Matrix ◽  
Epstein Barr Virus ◽  
Cellular Proteins ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Leader Protein ◽  
Epstein Barr ◽  
Self Association

ABSTRACT Self-association of viral proteins is important for many of their functions, including enzymatic, transcriptional, and transformational activities. Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) contains various numbers of W1W2 repeats and a unique carboxyl-terminal Y1Y2 domain. It was reported that EBNA-LP associates with a variety of cellular proteins and plays a critical role in EBV-induced transformation. We report here that EBNA-LP self-associates in vivo and the domain responsible for the homotypic association is a multifunctional domain mediating nuclear localization, nuclear matrix association, and EBNA-2-dependent coactivator function of the protein. Our conclusions are based on the following observations. (i) EBNA-LP interacts with itself or its derivatives in the yeast two-hybrid system. (ii) A purified chimeric protein consisting of glutathione S-transferase fused to EBNA-LP specifically formed complexes with EBNA-LP transiently expressed in COS-7 cells. (iii) When Flag epitope-tagged EBNA-LP with either one or two W1W2 repeats and EBNA-LP containing four W1W2 repeats were coexpressed in COS-7 cells, the latter was specifically coimmunoprecipitated with the former. (iv) Mutational analyses of EBNA-LP with deletion mutants revealed that the region between codons 19 and 39 (relative to the first amino acid residue of the W2 domain) is essential for self-association of the protein. The mapped region almost completely overlaps with CR2 and CR3, regions conserved among a subset of primate γ-herpesviruses and critical for EBNA-2-dependent coactivator function. Amino acid substitutions in CR2 alone abolished the ability of the protein to self-interact. This laboratory previously reported that CR2 is also responsible for nuclear localization and nuclear matrix association (A. Yokoyama, Y. Kawaguchi, I. Kitabayashi, M. Ohki, and K. Hirai, Virology 279:401–413, 2001). (v) Sucrose gradient sedimentation showed that amino acid substitutions in CR2 reduced the ability of the protein to form protein complexes in B cells. These results suggest that self-association of EBNA-LP may be important for its various functions and interactions of the protein with multiple cellular proteins.

scholarly journals Epstein-Barr Virus Nuclear Antigen Leader Protein Coactivates EP300

2018 ◽  
Vol 92 (9) ◽  
Author(s):  
Chong Wang ◽  
Hufeng Zhou ◽  
Yong Xue ◽  
Jun Liang ◽  
Yohei Narita ◽  
...  
Keyword(s):  
Transcription Factors ◽  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Transcription Activator ◽  
Lymphoproliferative Diseases ◽  
Barr Virus ◽  
Leader Protein ◽  
Epstein Barr

ABSTRACTEpstein-Barr virus nuclear antigen (EBNA) leader protein (EBNALP) is one of the first viral genes expressed upon B-cell infection. EBNALP is essential for EBV-mediated B-cell immortalization. EBNALP is thought to function primarily by coactivating EBNA2-mediated transcription. Chromatin immune precipitation followed by deep sequencing (ChIP-seq) studies highlight that EBNALP frequently cooccupies DNA sites with host cell transcription factors (TFs), in particular, EP300, implicating a broader role in transcription regulation. In this study, we investigated the mechanisms of EBNALP transcription coactivation through EP300. EBNALP greatly enhanced EP300 transcription activation when EP300 was tethered to a promoter. EBNALP coimmunoprecipitated endogenous EP300 from lymphoblastoid cell lines (LCLs). EBNALP W repeat serine residues 34, 36, and 63 were required for EP300 association and coactivation. Deletion of the EP300 histone acetyltransferase (HAT) domain greatly reduced EBNALP coactivation and abolished the EBNALP association. An EP300 bromodomain inhibitor also abolished EBNALP coactivation and blocked the EP300 association with EBNALP. EBNALP sites cooccupied by EP300 had significantly higher ChIP-seq signals for sequence-specific TFs, including SPI1, RelA, EBF1, IRF4, BATF, and PAX5. EBNALP- and EP300-cooccurring sites also had much higher H3K4me1 and H3K27ac signals, indicative of activated enhancers. EBNALP-only sites had much higher signals for DNA looping factors, including CTCF and RAD21. EBNALP coactivated reporters under the control of NF-κB or SPI1. EP300 inhibition abolished EBNALP coactivation of these reporters. Clustered regularly interspaced short palindromic repeat interference targeting of EBNALP enhancer sites significantly reduced target gene expression, including that of EP300 itself. These data suggest a previously unrecognized mechanism by which EBNALP coactivates transcription through subverting of EP300 and thus affects the expression of LCL genes regulated by a broad range of host TFs.IMPORTANCEEpstein-Barr virus was the first human DNA tumor virus discovered over 50 years ago. EBV is causally linked to ∼200,000 human malignancies annually. These cancers include endemic Burkitt lymphoma, Hodgkin lymphoma, lymphoma/lymphoproliferative disease in transplant recipients or HIV-infected people, nasopharyngeal carcinoma, and ∼10% of gastric carcinoma cases. EBV-immortalized human B cells faithfully model key aspects of EBV lymphoproliferative diseases and are useful models of EBV oncogenesis. EBNALP is essential for EBV to transform B cells and transcriptionally coactivates EBNA2 by removing repressors from EBNA2-bound DNA sites. Here, we found that EBNALP can also modulate the activity of the key transcription activator EP300, an acetyltransferase that activates a broad range of transcription factors. Our data suggest that EBNALP regulates a much broader range of host genes than was previously appreciated. A small-molecule inhibitor of EP300 abolished EBNALP coactivation of multiple target genes. These findings suggest novel therapeutic approaches to control EBV-associated lymphoproliferative diseases.

scholarly journals Identification of protein kinases responsible for phosphorylation of Epstein–Barr virus nuclear antigen leader protein at serine-35, which regulates its coactivator function

2003 ◽  
Vol 84 (12) ◽  
pp. 3381-3392 ◽  
Author(s):  
Kentaro Kato ◽  
Akihiko Yokoyama ◽  
Yukinobu Tohya ◽  
Hiroomi Akashi ◽  
Yukihiro Nishiyama ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Leader Protein ◽  
Epstein Barr

scholarly journals The CR4 region of EBNA2 confers viability of Epstein–Barr virus-transformed B cells by CBF1-independent signalling

2006 ◽  
Vol 87 (11) ◽  
pp. 3169-3176 ◽  
Author(s):  
Kristina Grabusic ◽  
Sabine Maier ◽  
Andrea Hartmann ◽  
Anja Mantik ◽  
Wolfgang Hammerschmidt ◽  
...  
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Cell Transformation ◽  
Mutant Virus ◽  
Nuclear Antigen ◽  
Population Doubling ◽  
Population Doubling Time ◽  
Barr Virus ◽  
Epstein Barr ◽  
Cell Densities

The Epstein–Barr virus (EBV) nuclear antigen 2 (EBNA2) gene product is the key regulator of the latent genes of EBV and essential for EBV-mediated transformation of human primary B cells. Viral mutants were constructed carrying a deletion of the EBNA2 conserved region 4 (CR4). Primary resting B cells infected with the ΔCR4-EBNA2 mutant virus were dramatically impaired for B cell transformation. Lymphoblastoid cell lines (LCLs) established with this mutant EBV revealed a prolonged population doubling time when cells were cultivated at low cell densities, which are not critical for wild-type-infected cells. Low-level spontaneous cell death occurred when the cells were cultivated at suboptimal cell densities. The phenotype of B cells and LCLs infected with the ΔCR4-EBNA2 mutant virus indicated that the CR4 region of EBNA2 specifically contributes to the viability of the cells rather than affecting cell division rates.

scholarly journals Intron Retention May Regulate Expression of Epstein-Barr Virus Nuclear Antigen 3 Family Genes

1999 ◽  
Vol 73 (2) ◽  
pp. 1195-1204 ◽  
Author(s):  
Norbert Kienzle ◽  
David B. Young ◽  
Daphne Liaskou ◽  
Marion Buck ◽  
Sonia Greco ◽  
...  
Keyword(s):  
Protein Expression ◽  
Epstein Barr Virus ◽  
Intron Retention ◽  
Fine Tuning ◽  
Nuclear Antigen ◽  
Cell Fractionation ◽  
Flag Epitope ◽  
Barr Virus ◽  
Epstein Barr ◽  
The Individual

ABSTRACT The nuclear antigen 3 family genes (EBNA-3, EBNA-4, and EBNA-6) of Epstein-Barr virus (EBV) are important for EBV-induced immortalization and survival of B lymphocytes. However, little is known about how the expression of these genes is regulated. Each of the EBNA-3, EBNA-4, and EBNA-6 genes consists of two exons separated by a small intron. Reverse transcriptase PCR assays revealed that the vast majority of the EBNA-3, EBNA-4, and EBNA-6 mRNA, expressed in transfected and EBV-infected B cells, retained intron sequences. Northern blot and S1 protection assays confirmed that most of the EBNA-3 mRNA contained intron. Examination of deletion mutants of EBNA-3 indicated that the EBNA-3 protein was not necessary for intron retention and that there was no splicing silencing element encoded in the EBNA-3 mRNA. Cell fractionation and RNA gradient analysis revealed that the unspliced EBNA 3 family mRNAs were transported into the cytoplasm and associated with the polysomes. However, Western blot analysis of FLAG-epitope tagged EBNA-3 gave no indication of the presence of splice variant protein forms of EBNA-3. In contrast, transiently transfected cells expressing EBNA-3 revealed a sixfold increase in EBNA-3 protein expression from the genomic EBNA-3 gene compared to EBNA-3 cDNA. These data show that the intronic sequences can influence EBNA-3 protein expression and suggest that intron retention may provide a means for the fine-tuning of expression of the individual EBNA 3 family genes.

scholarly journals Functional Interaction of Nuclear Factor Y and Sp1 Is Required for Activation of the Epstein-Barr Virus C Promoter

2003 ◽  
Vol 77 (2) ◽  
pp. 821-829 ◽  
Author(s):  
Cecilia Boreström ◽  
Henrik Zetterberg ◽  
Kristian Liff ◽  
Lars Rymo
Keyword(s):  
Epstein Barr Virus ◽  
Cell Transformation ◽  
Dominant Negative ◽  
Nuclear Antigen ◽  
Nuclear Factor ◽  
Barr Virus ◽  
Nuclear Factor Y ◽  
Epstein Barr ◽  
B Lymphoid ◽  
Virus C

ABSTRACT Two Epstein-Barr virus (EBV) latent cycle promoters, Wp and Cp, are activated sequentially during virus-induced transformation of primary B lymphocytes. Immediately postinfection, viral transcription initiates from Wp, leading to expression of EBV nuclear antigen 2 (EBNA2) and EBNA5. Within 36 h, there is a switch in promoter usage from Wp to the upstream Cp, which leads to expression of EBNA1 to EBNA6. EBNA2 appears to be required for the Wp-to-Cp switch, but the switching mechanism is not fully understood at the molecular level. In a previous investigation we showed that there is an EBNA2-independent activity of reporter constructs containing deletion fragments of Cp in B-lymphoid cell lines, and we demonstrated that Cp activity is highly dependent on several cellular transcription factors, including nuclear factor Y (NF-Y) and Sp1. In the present work, we analyzed the effect of NF-Y on Cp activity in greater detail. We demonstrate that (i) a dominant negative analogue of NF-Y abolishes Cp activity, (ii) NF-Y and Sp1 costimulate Cp, and (iii) the oriPI-EBNA1-induced transactivation of Cp requires concomitant expression of NF-Y and Sp1, although additional factors seem necessary for optimal activation. Furthermore, using the lymphoblastoid cell line EREB2-5, in which EBNA2 function is regulated by estrogen, we demonstrate that inactivation of EBNA2 results in decreased expression of NF-Y and down-regulation of Cp. On reconstitution of the EBNA2 function, the cells enter the cell cycle, NF-Y levels increase, and a concomitant Wp-to-Cp switch occurs. Taken together, our results suggest that NF-Y is essential for Cp activation and that up-regulation of NF-Y may contribute to a successful Wp-to-Cp switch during B-cell transformation.

scholarly journals Antigenic and Sequence Variation in the C-Terminal Unique Domain of the Epstein-Barr Virus Nuclear Antigen EBNA-1

Virology ◽  
1995 ◽  
Vol 208 (2) ◽  
pp. 521-530 ◽  
Author(s):  
Mark N. Wrightham ◽  
James P. Stewart ◽  
Nusrat J. Janjua ◽  
Stuart De V. Pepper ◽  
Clare Sample ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Sequence Variation ◽  
Nuclear Antigen ◽  
Barr Virus ◽  
Epstein Barr ◽  
Unique Domain

scholarly journals Epstein-Barr Virus Nuclear Antigen Leader Protein Induces Expression of Thymus- and Activation-Regulated Chemokine in B Cells

2004 ◽  
Vol 78 (8) ◽  
pp. 3984-3993 ◽  
Author(s):  
Mikiko Kanamori ◽  
Shinya Watanabe ◽  
Reiko Honma ◽  
Masayuki Kuroda ◽  
Shosuke Imai ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Critical Role ◽  
Nuclear Antigen ◽  
Viral Gene ◽  
Barr Virus ◽  
Leader Protein ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) plays a critical role in transformation of primary B lymphocytes to continuously proliferating lymphoblastoid cell lines (LCLs). To identify cellular genes in B cells whose expression is regulated by EBNA-LP, we performed microarray expression profiling on an EBV-negative human B-cell line, BJAB cells, that were transduced by a retroviral vector expressing the EBV EBNA-LP (BJAB-LP cells) and on BJAB cells that were transduced with a control vector (BJAB-vec cells). Microarray analysis led to the identification of a cellular gene encoding the CC chemokine TARC as a novel target gene that was induced by EBNA-LP. The levels of TARC mRNA expression and TARC secretion were significantly up-regulated in BJAB-LP compared with BJAB-vec cells. Induction of TARC was also observed when a subline of BJAB cells was converted by a recombinant EBV. Among the EBV-infected B-cell lines with the latency III phenotype that were tested, the LCLs especially secreted significantly high levels of TARC. The level of TARC secretion appeared to correlate with the level of full-length EBNA-LP expression. These results indicate that EBV infection induces TARC expression in B cells and that EBNA-LP is one of the viral gene products responsible for the induction.

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